JP2019515693A5 - - Google Patents

Description

Preferably, the one or more nucleotide sequences for expression in the plant are adapted for expression in the plant and alter or modify plant traits.
In certain preferred embodiments, one or more of the nucleotide sequences for expression in plants comprises a nucleotide sequence encoding a protein. In one preferred embodiment, the nucleotide sequence encoding the protein comprises a nucleotide sequence, or a fragment or variant thereof represented by SEQ ID NO: 38～46,76 or 7 8,.

In certain preferred embodiments, one or more of the nucleotide sequences suitable for expression in plants is a non-protein encoding nucleotide sequence. Preferably, said non-protein encoding nucleotide sequence comprises one or more small RNA nucleotide sequences. In one preferred embodiment, includes one or more small RNA nucleotide sequence, said nucleotide sequence for expression, SEQ ID NO: 12～26,64～66,80～81,83～92,94, or at 96 to 97 Contains the nucleotide sequence shown, or a fragment or variant thereof.

In a preferred embodiment, the one or more nucleotide sequences for expression in a plant comprise one or more selectable marker nucleotide sequences. In a preferred embodiment, the selectable marker nucleotide sequence comprises the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs: 38-46, or the nucleotide sequence set forth in SEQ ID NO: 115 , or a fragment or variant thereof.

Preferably, the regulatory nucleotide sequence comprises one or more promoter sequences. In one preferred embodiment, the promoter nucleotide sequence comprises a nucleotide sequence, or a fragment or variant thereof represented by SEQ ID NO: 4～7,67,73,74,76 or 7 8,.

Preferably, the control sequence comprises one or more terminator sequences. In one preferred embodiment, the terminator nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NOs: 8-11, 102 , 104 , 107 , or 108 , or a fragment or variant thereof.

In some preferred embodiments, the flanking sequence includes a restriction digestion site. In certain particularly preferred embodiments, one or more of the flanking sequences is a nucleotide sequence set forth in SEQ ID NO: 98 , 99 , 105 , 106 , 111 , 112 , 113 , 114 , 116 , or 117 , or a fragment or mutation thereof. Including the body.

In certain particularly preferred embodiments, the recombinant gene constructs of this embodiment, the nucleotide sequences or SEQ ID NO are shown in SEQ ID NO: 1～35,49,51～56,66～68,71～92 or 94-97, The nucleic acid which codes the amino acid sequence shown by 38-46, or its fragment | piece or variant is included.

In some particularly preferred embodiments where the recombinant gene construct comprises border sequences, the gene construct comprises the nucleotide sequence set forth in SEQ ID NOs: 1-35, 49, 51-66, 81, 94, or 97 , and / or The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NOs: 38 to 46, or a fragment or variant thereof is included.

In a third aspect, the present invention provides a genetic construct produced according to the method of the second aspect. In a particularly preferred embodiment, the gene construct is represented by the nucleotide sequence or SEQ ID NO: 38-46, SEQ ID NO: 1～35,49,51～56,66～68,71～92 or 94-97, It includes a nucleic acid encoding an amino acid sequence, or a fragment or variant thereof.

The figure which shows the schematic of the gene construct of this invention, and the vector (pIntR2) of this invention containing the said gene construct. The nucleotide sequence of this gene construct is shown in SEQ ID NO: 1. The figure which shows the schematic of the vector of this invention containing the gene construct of this invention, and the said gene construct. The figure which shows the schematic of the vector of this invention containing the gene construct of this invention, and the said gene construct. FIG. 6 shows the results of transient transformation of tomato mesophyll protoplasts using the pRbcS3C: sGFP: tRbcS3C construct and p35S: sGFP: tNOS as a control. The figure which shows the result of the expression of pRbcS3C: sGFP: tRbcS3C in the tomato leaf in a vascular tissue and a stoma. Comparison of GFP expression driven by promoter-terminator pairs belonging to tomato ACTIN (Act7), CYCLOPHILIN (CyP40) and UBIQUITIN (Ubi3) genes by transient expression in Nicotiana benthamiana leaves FIG. Same as above. Same as above. Tomato ACTIN (Act7; left column), CaMV 35S (middle column) and RUBISCO subunit 3C (RbcS3C) gene (right column) due to transient expression in agroinfiltrated N. benthamiana leaves The figure which shows the comparison of the GFP expression driven by the promoter-terminator pair which belongs. FIG. 3 shows the results of regeneration from tomato cotyledons transformed with an intragenic pRbcS3C: GS1G245C: tRbcS3C construct on selective 1 mg / L GA medium for 2 weeks; the two plates on the left are Control cotyledons that were not co-incubated with Agrobacterium harboring The figure which shows the result of the initial reproduction | regeneration from the tomato cotyledon transformed using the gene pRbcS3C: GS1G245C: tRbcS3C construct on selective 1 mg / L GA culture medium for 4 weeks. The figure which shows the result of using the tomi-derived amiRNA construct for targeting the cucumber mosaic virus sequence. Shown is a dual LUC assay after agroinfiltration of N. benthamiana leaves. N = 6; error bars represent the standard error of the mean value. Same as above. The figure which shows CMV disease symptom expression in the expression plant of five wild type versus five ami10 (sequence number 15). A: CMV symptom expression in wild type (upper panel) versus ami10 (lower panel) plants 3 weeks after CMV inoculation. B: CMV symptom expression in wild type (left) versus ami10 (right) 3 weeks after CMV inoculation. The figure which shows the quantification by qRT-PCR of the CMV viral load in the expression plant of five wild type versus five ami10 (sequence number 15). The relative expression ratio was calculated based on the geometric mean of the relative ratios of the two reference genes ACTIN and GAPDH. Same as above. Shown is the process of designing an RNAi construct having the nucleotide sequence shown in SEQ ID NO: 18, using the tomato (cultivar Moneymaker) sequence, used and derived together by bioinformatics to create SEQ ID NO: 18, And each plant-derived sequence is at least 20 nucleotides in length. Same as above. Same as above. Same as above. FIG. 5 shows the results of using tomato-derived RNAi constructs to target the cucumber mosaic virus sequence. Shown are the results of a double LUC assay after agroinfiltration of N. benthamiana leaves. N = 6; error bars represent standard error of the mean; t-test showed a highly significant difference. FIG. 1 shows the sequence of the genetic construct (SEQ ID NO: 1) contained in the basic intragenic cloning vector pIntR2 illustrated in FIG. 1 and includes first and second boundaries including Agrobacterium RB and LB Figure 2 shows the nucleotide sequence (bold); tomato RbcS3C promoter and terminator (underlined); and restriction enzyme sites (bold) used for insertion of genes and additional in-gene expression cassettes. Note that the first border nucleotide sequence (RB sequence) is represented at the 5 'end of the sequence and the second border nucleotide sequence (LB sequence) is represented at the 3' end of the sequence. Agrobacterium-mediated T-DNA insertion mutant plant (med18) virus resistance (A); and tomato MED18 suppression using tomato-derived amiRNA sequences. Same as above. The figure which shows sequence number 1-66, 68, 71-72, 75, 77, 80, 83-89, 93, and 95 in FASTA format. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. FIG. 6 shows the nucleotide sequence and structure of pIntrA (SEQ ID NO: 67), a preferred cloning construct of the present invention. BbvCI restriction enzyme site (SEQ ID NO: 98 ); SphI restriction enzyme site (SEQ ID NO: 99 ); RB (SEQ ID NO: 100 ); LB (SEQ ID NO: 101 ); HpaI restriction enzyme site; PmlI restriction enzyme site; The partial actin 7 (PARTIAL ACTIN7) promoter (SEQ ID NO: 102 ) and partial actin 7 (PARTIAL ACTIN7) terminator (SEQ ID NO: 103 ) are represented by highlighting and / or underlining. . The figure which shows the nucleotide sequence (sequence number 69) and structure of a structure containing the selectable marker gene (nptII) which does not belong to a plant or are not derived for using for the co-transformation which combined the gene construct of this invention. RB; LB, nptII selectable marker; double 35S promoter; no terminator, ANT1 Solanum chilense anthocyanin gene; tomato ACTIN7 promoter; and tomato RbcS3C terminator are represented by highlighting and / or underlining . Same as above. The nucleotide sequence of a vector of the invention comprising a preferred gene construct of the invention together with a further gene construct comprising a selectable marker gene (nptII) that does not belong to or not derived from a plant for use in cotransformation according to the invention ( SEQ ID NO: 70) and diagram showing the structure. HpaI restriction enzyme site; PmlI restriction enzyme site; RB; LB, nptII selection marker; visual selection ANT1 marker, and partial ACTIN promoter and terminator are represented by highlighting and / or underlining. Same as above. Same as above. Same as above. Same as above. FIG. 7 shows pSbiUbi1 (SEQ ID NO: 73), a preferred cloning construct of the present invention comprising a Ubi1 promoter and terminator from Sorghum bicolor, a CTGCAG PstI restriction enzyme site; and a ggcGCC SfoI restriction enzyme site. The Ubi1 promoter and terminator derived from Sobic 004G050000 (SEQ ID NO: 104 ); the CTGCAG PstI restriction enzyme site (SEQ ID NO: 105 ); and the ggcGCC SfoI restriction enzyme site (SEQ ID NO: 106 ) are tabulated by highlighting and / or underlining. Is done. Same as above. PSbiU74, a preferred cloning construct of the present invention comprising the Ubi2 promoter from Sorghum bicolor; the Ubi1 terminator from Sorghum bicolor; the CTGCAG PstI restriction enzyme site; and the ggcGCC SfoI restriction enzyme site. ). Sobic. Ubi2 promoter derived from 004G049900 (SEQ ID NO: 107 ) and Sobic. The Ubi1 terminator from 004G050000; and the CTGCAG PstI restriction enzyme site; the ggcGCC SfoI restriction enzyme site are represented by highlighting and / or underlining. Same as above. An oryza sativa APX promoter and terminator; and a multiple cloning site of gagcTCCGGATTATAa consisting of SacI or Eco53kI and blunt cutter PsiI; GAACGt and cGATC: XmnI restriction enzyme site, the preferred cloning construct of the present invention, XOsAP ). APX promoter (SEQ ID NO: 108 ); APX terminator (SEQ ID NO: 109 ); gagcTCCGGATTATAa multiple cloning site (SEQ ID NO: 110 ) consisting of SacI or Eco53kI and blunt cutter PsiI; GAACCGt (SEQ ID NO: 111 ) and cGATC (SEQ ID NO: 112 ): XmnI restriction enzyme sites are represented by highlighting and / or underlining. The figure which shows the tomato plant which expresses sequence number 69 which shows the raise of anthocyanin level (purple stem, a root, a vein, and a part of leaf). FIG. 70 shows a tomato plant (left) cotransformed with the vector of the invention shown in SEQ ID NO: 69, showing strong anthocyanin production compared to a control tomato plant (right). Asian rice (ORyza sativa) DREB1A gene; Asian rice (Oryza sativa) ACTIN1 promoter, and Asian rice (Oryza sativa) DREB1A terminator nucleotide sequence of the present invention ACTIN1: DREB1A: DREB1A gene construct showing SEQ ID NO: 78 The gene construct further includes NheI and PmlI restriction digestion sites for excision and cloning. NheI (SEQ ID NO: 113 ) and PmlI (SEQ ID NO: 114 ) restriction sites; DREB1A coding sequence (SEQ ID NO: 115 ); and GTGTT sequences added to the 3 ′ end of the DREB1A coding sequence are underlined and / or underlined. Is represented by FIG. 9 shows the NCED3: DREB1A: NCED3 gene construct (SEQ ID NO: 79) of the present invention comprising the nucleotide sequence of the Asian rice (Oryza sativa) DREB1A gene; and the Asian rice (Oryza sativa) NCED3 promoter and terminator. Additional TGC (SEQ ID NO: 116 ) and GCA (SEQ ID NO: 117 ) nucleotides; NCED3 promoter (SEQ ID NO: 118 ); and NCED3 terminator (SEQ ID NO: 119 ); and DREB1A coding sequences are represented by highlighting and / or underlined use. Is done. The figure which shows the reproduction | regeneration of the rice callus transformed by ACTIN1: DREB1A: DREB1A (left) or NCED3: DREB1A: NCED3 (right) on the culture medium containing 100 mM NaCl. The figure which shows the ami11-IT1 plant inoculated with CMV and the wild type control tomato plant inoculated with CMV. All wild-type plants show “slight” symptoms in the new growth (right side). Most ami11-I plants are asymptomatic (left side). FIG. 3 shows ELISA evaluation of CMV load in wild type, T1 unpaired and ami11-IT1 tomato plants. FIG. 5 shows ELISA evaluation of CMV load in wild type, T1 unpaired, and ami11-II T1 tomato plants. The figure which shows the evaluation of the severity of a CMV and plant height in ami11-I and ami11-II tomato plants. The figure which shows the fruit number and exemplary fruit form from the ami11-I and ami11-II strains infected with CMV. FIG. 6 shows the nucleotide sequence of a “double” anti-CMV amiRNA insert with tomato-derived anti-CMV ami10 and ami11, and an evaluation of the RNA targeting of the insert. The figure which shows the nucleotide sequence (sequence number 81) and structure of the preferable gene construct of this invention containing CMV amiRNA10 and amiRNA11. LB; Actin promoter; CMV amiRNA10 in Sly-miR156b; amiRNA11 in Sly-miR156a; Actin terminator; and RB are represented by emphasis / text color. The figure which shows the nucleotide sequence (sequence number 82) and structure of the preferable vector of this invention containing the gene construct shown in FIG. 35 in combination with the gene construct which has the selectable marker shown in FIG. The vector components are represented by emphasis / text color. Same as above. Same as above. Same as above. Same as above. Intragenic TSWV targeted amiRNA7 sequence (SEQ ID NO: 83); Evaluation of RNA targeting by this sequence using a dual LUC assay after agroinfiltration of N. benthamiana leaves (error bars are Represents the standard error of the mean); and a diagram showing an exemplary morphology of tomato plants transformed to express this sequence. The figure which shows the nucleotide sequence (sequence number 84-85) of sorghum-derived amiRNA (amiRNA3 and amiRNA6) which targets the conserved region of MDMV and SCMV, evaluation of RNA targeting by these sequences, and regeneration of sorghum plants. Successful transformants are expected to have the MDMV / SCMV resistance phenotype. The figure which shows the nucleotide sequence (sequence number 86-89) of sorghum origin amiRNA (amiRNA2, amiRNA4, amiRNA5, and amiRNA7) which targets JGMV, evaluation of RNA targeting by these sequences, and regeneration of a sorghum plant. Successful transformants are expected to have a JGMV resistance phenotype. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 90) and structure of a gene construct of the present invention comprising a sorghum Ubi1 promoter and terminator and three sorghum-derived amiRNAs that target JGMV (amiRNA4, amiRNA5, and amiRNA2). The components of the construct are represented by text color. Same as above. Same as above. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 91) and structure of a preferred gene construct of the present invention comprising a sorghum Ubi2 promoter and a sorghum Ubi1 terminator, and three sorghum-derived amiRNAs that target JGMV (amiRNA4, amiRNA5, and amiRNA2). The components of the construct are represented by text color. Same as above. The figure which shows the nucleotide sequence (sequence number 92) of rice origin RTSV amiRNA1. FIG. 10 shows the design of a tomato-derived hairpin RNAi construct (SEQ ID NO: 94) that targets TSWV. The complete nucleotide sequence of the RNAi vector is shown in SEQ ID NO: 95. Same as above. Same as above. Same as above. Same as above. Same as above. Evaluation of RNA targeting by the construct shown in FIG. 43; exemplary phenotype of tomato plants transformed with the construct shown in FIG. 43; and compared to wild-type tomato plants when loaded with TSWV The figure which shows the TSWV load in the tomato plant transformed using the construct shown in FIG. Targeting MED18 by tomi-derived amiRNA27; expression of amiRNA27 and MED18 in transformed tomato plants compared to wild-type control; and CMV loading in wild-type compared to amiRNA27-transformed plant (labeled med18) . Same as above. Results of leaf shedding P. syringae assay in control (labeled W or WT) compared to amiRNA27 transformed (labeled A or MED18) tomato plants; and P. syringae The figure which shows the abundance of P. syringae in the control | contrast compared with the amiRNA27 transformed line | wire measured by Gyrase qPCR. Same as above. The figure which shows the reproduction | regeneration and growth of the rice plant transformed using ACTIN1: DREB1A: DREB1A on the culture medium containing 100 mM NaCl. The figure which shows the reproduction | regeneration and growth of the rice plant transformed by using NCED3: DREB1A: NCED3 on the culture medium containing 100 mM NaCl. The figure which shows the comparison of the form of the tomato plant transformed using the tomato origin amiRNA27 compared with a wild type control strain | stump | stock. FIG. 5 shows the nucleotide sequence of tomi-derived amiRNA6 targeting MED25; evaluation of targeting of MED25 by amiRNA6; and the expression of amiRNA6 and MED25 in tomato lines transformed with amiRNA6 compared to wild-type control lines. Shows the expression of anthocyanin in the reproduction rice plants transformed (right) with and Lee Ne derived construct; expression (left) of the anthocyanins in tomato lines transformed with the construct of SEQ ID NO: 69 Figure. Nucleotide sequence (SEQ ID NO: 97 ) and structure of a tomato-derived hairpin RNAi construct targeting the tomato gene encoding the gamma subunit (GGB1) of the type B heterotrimeric G protein; and the construct and SEQ ID NO: 69 FIG. 2 shows an exemplary transformed tomato plant cotransformed with a construct that also expresses anthocyanins. The figure which shows the developing rice plant produced by the fine particle gun using the rice origin RNAi construct which targets rice BADH2. Successful transformants are expected to have a fragrant phenotype.

SEQ ID NO: 18 Nucleotide sequence of tomato-derived RNAi construct targeting CMV SEQ ID NO: 19 Nucleotide sequence of fragment of SEQ ID NO: 18 highly similar to nucleotides 751-896 of CMV K segment 1 replicase SEQ ID NO: 20 of CMV K segment 1 replicase The nucleotide sequence of the fragment of SEQ ID NO: 18 highly similar to nucleotides 1235 to 1358 SEQ ID NO: 21 The nucleotide sequence of the fragment of SEQ ID NO: 18 highly similar to nucleotides 250 to 375 of CMV K segment 3 orf2 (coat protein) Tomato yellow spot virus (Tomato spotted wilt)
nucleotide) of tomato-derived RNAi construct targeting (TSWV) SEQ ID NO: 23 TSWV QLD1 segment L The nucleotide sequence of a fragment of SEQ ID NO: 22 highly similar to nucleotides 1918-2155 of RDRP SEQ ID NO: 24 TSWV QLD1 segment L RDRP The nucleotide sequence of the fragment of SEQ ID NO: 22 highly similar to nucleotides 8429-8639 of SEQ ID NO: 25 TSWV QLD1 segment The nucleotide sequence of the fragment of SEQ ID NO: 22 highly similar to nucleotides 187-360 of Morf SEQ ID NO: 26 TSWV QLD1 segment Nucleotide sequence of a fragment of SEQ ID NO: 22 that is highly similar to nucleotides 297-510 of Morf2 SEQ ID NO: 27 Tomato betaine aldehyde dehydrogenase (B DH) Nucleotide sequence of cDNA (gi20932342) SEQ ID NO: 28 Nucleotide sequence of tomato sorbitol dehydrogenase (SDH) cDNA (gi78183415) SEQ ID NO: 29 Nucleotide sequence of tomato osmotin CDS (gi460400210) SEQ ID NO: 30 Tomato glutamine synthetase (GTS) cDNA (gi460409535) Nucleotide sequence SEQ ID NO: 31 Nucleotide sequence of tomato phytoene desaturase cDNA (gi51275322) SEQ ID NO: 32 Nucleotide sequence of tomato 5-enolpyruvyl-3-phosphoshikimate cDNA (gi822202668) SEQ ID NO: 33 Nucleotide of tomato acetolactate synthase cDNA (gi723680771) Sequence No. 34 Tomato Nucleotide sequence of Protoporphyrinogen Oxidase cDNA (gi723658549) SEQ ID NO: 35 Nucleotide sequence of Solanum chilense anthocyanin 1 (ANT1) cDNA (gi126653934) SEQ ID NO: 36 Nucleotide sequence of tomato chlorophyll synthase cDNA (gi460401624) SEQ ID NO: 37 Nucleotide sequence of barnase suicide construct codon-optimized for Solanum expression with an intron derived from the potato ST-LS1 gene SEQ ID NO: 38 amino acid sequence of betaine aldehyde dehydrogenase encoded by SEQ ID NO: 27 Tomato sorbitol dehydrogenase protein encoded by SEQ ID NO: 28 Amino acid sequence of tomato osmotin protein encoded by SEQ ID NO: 29 amino acid sequence of tomato glutamine synthetase protein encoded by SEQ ID NO: 30 SEQ ID NO: 42 tomato phytoene desaturase protein encoded by SEQ ID NO: 31 Amino acid sequence of tomato 5-enolpyruvyl-3-phosphoshikimate protein encoded by SEQ ID NO: 32 SEQ ID NO: 44 amino acid sequence of tomato acetolactate synthase protein encoded by SEQ ID NO: 33 Amino acid sequence of tomato ProtOx protein encoded by number 34 SEQ ID NO: 46 Solanum cilense encoded by SEQ ID NO: 35 (Sol) num chilense) amino acid sequence of anthocyanin 1 protein SEQ ID NO: 47 nucleotide sequence of the basic intragenic cloning vector pIntR2 illustrated in FIG. 1 SEQ ID NO: 48 nucleotide sequence of the vector “pIntR2 GS1 G245C CML18” of the present invention SEQ ID NO: 49 native GS1 Nucleotide sequence of glutamine synthetase 1 (GS1) G245C marker gene operably linked to promoter and terminator sequences SEQ ID NO: 50 Nucleotide sequence of modified pArt27 backbone of the invention SEQ ID NO: 51 Tomato GS1 encoding CD245 of G245T protein Nucleotide sequence SEQ ID NO: 52 CDS nucleotide sequence of tomato GS1 C745T CDS encoding H249Y protein No. 53 Nucleotide sequence of tomato GS1 promoter SEQ ID NO: 54 Nucleotide sequence of tomato GS1 terminator SEQ ID NO: 55 Nucleotide sequence of tomato phytoene desaturase promoter SEQ ID NO: 56 Nucleotide sequence of tomato phytoene desaturase terminator SEQ ID NO: 57 Nucleotide sequence of tomato acetolactate synthase promoter 58 Nucleotide sequence of tomato acetolactate synthase terminator SEQ ID NO: 59 Nucleotide sequence of tomato 5-enolpyruvyl shikimate-3-phosphate synthase promoter SEQ ID NO: 60 Nucleotide sequence of tomato 5-enol pyruvyl shikimate-3-phosphate synthase terminator Sequence SEQ ID NO: 61 Nucleotide sequence of tomato ProtOx promoter SEQ ID NO: 62 Tomato Pro Nucleotide sequence of tOx terminator SEQ ID NO: 63 Nucleotide sequence of intragenic cloning vector pIntR 2 (SEQ ID NO: 1) which is removed upon digestion with PmlI and PciI restriction enzymes and facilitates ligation of nucleotide sequence to pIntR 2 SEQ ID NO: 64 derived from tomato Nucleotide sequence of the MED18 gene (gi | 723704094 | ref | XM — 010323502.1)
SEQ ID NO: 65 Nucleotide sequence of amiRNA sequence targeting tomato MED18 (MED18ami3) SEQ ID NO: 66 Nucleotide sequence of amiRNA sequence targeting tomato MED18 (MED18ami27) SEQ ID NO: 67 Nucleotide sequence of basic intragenic cloning construct of pIntrA No. 68 Nucleotide sequence of a removable sequence having a restriction digestion site of pIntrA SEQ ID NO: 69 comprising a selectable marker gene (nptII) that does not belong to or does not originate from a plant used in co-transformation in conjunction with the gene construct of the present invention Nucleotide sequence of the construct SEQ ID NO: 70 Additional gene used in co-transformation according to the present invention, including a selectable marker gene (nptII) that does not belong to or is not derived from a plant Nucleotide sequence of a vector of the invention comprising a preferred gene construct of the invention together with a construct SEQ ID NO: 71 Nucleotide sequence of a portion of the first border sequence in a particular preferred gene construct of the invention SEQ ID NO: 72 of the invention The nucleotide sequence of a portion of the second border sequence in certain preferred gene constructs SEQ ID NO: 73 The nucleotide sequence of pSbiUbi1 SEQ ID NO: 74 The nucleotide sequence of pSbiUbi2 SEQ ID NO: 75 The nucleotide sequence of the spacer of the pSbiUbi1 and pSbiUbi2 cloning sites SEQ ID NO: 76 pOsaAPX construct The nucleotide sequence of SEQ ID NO: 77 The nucleotide sequence of the spacer of the pOsaAPX cloning site SEQ ID NO: 78 The nucleotide sequence of the rice ACTIN1: DREB1A: DREB1A construct SEQ ID NO: 79 Nucleotide sequence of rice NCED3: DREB1A: NCED3 construct SEQ ID NO: 80 Nucleotide sequence of double anti-CMV amiRNA insert from tomato SEQ ID NO: 81 Nucleotide sequence of intragenic tomato-derived construct comprising SEQ ID NO: 80 Vector comprising SEQ ID NO: 82 Nucleotide sequence of SEQ ID NO: 83 nucleotide sequence of anti-TSVV amiRNA7 from tomato SEQ ID NO: 84 nucleotide sequence of sorghum derived amiRNA3 targeting conserved regions of MDMV and SCMV SEQ ID NO: 85 sorghum derived amiRNA6 targeting conserved regions of MDMV and SCMV Nucleotide sequence of SEQ ID NO: 86 Nucleotide sequence of amiRNA2 derived from sorghum targeting JGMV SEQ ID NO: 87 derived from sorghum targeting JGMV Nucleotide sequence of miRNA4 SEQ ID NO: 88 Nucleotide sequence of sorghum-derived amiRNA5 targeting JGMV SEQ ID NO: 89 Nucleotide sequence of sorghum-derived amiRNA7 targeting JGMV SEQ ID NO: 90 Nucleotide sequence of sorghum-derived triple anti-JGMV amiRNA construct in pSbiUbi1 91 nucleotide sequence of sorghum-derived triple anti-JGMV amiRNA construct in pSbiUbi2 SEQ ID NO: 92 nucleotide sequence of rice-derived amiRNA1 targeting RTSV SEQ ID NO: 93 nucleotide sequence of vector comprising SEQ ID NO: 92 derived from tomato targeting TSWV Nucleotide sequence of hairpin RNAi SEQ ID NO: 95 nucleotide sequence of vector comprising SEQ ID NO: 94 SEQ ID NO: 96 MED25 Nucleotide sequence of tomato-derived amiRNA6 targeting tomato SEQ ID NO: 97 nucleotide sequence of tomato-derived hairpin RNAi construct targeting tomato gene encoding γ subunit (GGB1) of type B heterotrimeric G protein SEQ ID NO: 98 BbvCI restriction Nucleotide sequence of the enzyme site SEQ ID NO: 99 Nucleotide sequence of the SphI restriction enzyme site SEQ ID NO: 100 Nucleotide sequence of the RB sequence SEQ ID NO: 101 Nucleotide sequence of the LB sequence SEQ ID NO: 102 Partial nucleotide sequence of the partial ACTIN7 promoter SEQ ID NO: 103 portion actin 7 (partial ACTIN7) terminator nucleotide sequence SEQ ID NO: 104 sorghum Ubi1 promoter and terminator nucleotide sequence SEQ ID NO: 05 PstI nucleotide sequence SEQ ID NO: 106 Sfol restriction sites of the nucleotide sequence SEQ ID NO: 107 sorghum Ubi2 promoter nucleotide sequence SEQ ID NO: 108 Nucleotide sequence SEQ ID NO: 110 multiple cloning of pOsaAPX nucleotide sequence SEQ ID NO: 109 Rice APX terminator rice APX promoter restriction sites The nucleotide sequence of the site SEQ ID NO: 111 The nucleotide sequence of the XmnI restriction site SEQ ID NO: 112 The nucleotide sequence of the XmnI restriction site SEQ ID NO: 113 The nucleotide sequence of the NheI restriction site SEQ ID NO: 114 The nucleotide sequence of the PmlI restriction site SEQ ID NO: 115 with the addition of 3′GTGTT rice DREB1A nucleotide sequence sequence coding SEQ ID NO: 116 FspI restriction site nucleotide sequence SEQ ID NO: 1 7 FspI Tomato derived nucleotide sequence SEQ ID NO: within 121 Sly-miR156a anti CMV AmiRNA10 in the restriction sites of the nucleotide sequence SEQ ID NO: 118 Rice NCED3 promoter nucleotide sequence SEQ ID NO: 119 Rice NCED3 terminator nucleotide sequence SEQ ID NO: 120 Sly-miR156b the nucleotide sequence of the primers shown in the nucleotide sequence SEQ ID NO: 122 to medium 149 herein from anti CMV amiRNA11 (detailed description)
The present invention is predicted based, at least in part, on the recognition that there is a need for genetic improvements in plants, where the introduction of nucleotide sequences that are not or cannot be derived from plants into plant genetic material is Avoided.

In certain embodiments, a nucleic acid "fragment", SEQ ID NO: 1～35,49,51～56,66～68,71～92, or less than 100% nucleotide sequence represented by 94 to 97, at least A nucleotide sequence comprising 20%, preferably at least 30%, more preferably at least 80% or even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%.

In a preferred embodiment, the fragment of the gene construct of the present invention has a nucleotide sequence of 10, 12, 15, 20, 30, 40 of SEQ ID NO: 1, 67, 73-74, 76, 81, 95 , or 97. 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, or 3000 contiguous nucleotides including.

In some embodiments, nucleic acid variants of SEQ ID NO 1～35,49,51～56,66～68,71～92, or 94-97 nucleotide sequence represented by at least 75%, 80%, 85 It includes isolated nucleic acids having%, 90% or 95%, 96%, 97%, 98% or 99% nucleotide sequence identity.

Referring to the examples, a preferred recombinant gene construct pIntR 2 of this aspect comprises 1110 base pairs adapted for insertion into plant genetic material, and the construct is for insertion or integration into plant genetic material. It will be appreciated that the cloning construct is designed to receive additional plant-derived nucleotide sequences. Similarly, the preferred recombinant gene construct pIntRA of this embodiment comprises 1787 base pairs adapted for insertion into plant genetic material, and this construct is further added for insertion or integration into plant genetic material. A cloning construct designed to receive a plant-derived nucleotide sequence. Further, it preferred recombinant gene construct of SEQ ID NO: 78,79,81, and 97 each include 2387,3369,2084, Contact and 3071 base pairs to accommodate insertion into genetic material of the plant.

In certain particularly preferred embodiments, the nucleotide sequence encoding the protein comprises a nucleotide sequence, or a fragment or variant thereof represented by SEQ ID NO: 38～46,76 or 7 8,.

In a particularly preferred embodiment, it comprises a small RNA sequence, wherein the non-coding nucleotide sequence in the expression, the nucleotide sequence, or a fragment or variant of SEQ ID NO: 12～26,80,81,83～92 or 94-97, Including the body.

In particularly preferred embodiments, the one or more selectable marker nucleotide sequences are represented by one or more of SEQ ID NOs: 27-35 or 115 , or fragments or variants thereof, or any one of SEQ ID NOs: 38-46. One or more nucleotide sequences each encoding the amino acid sequence to be encoded, or a fragment or variant thereof.

In a preferred embodiment, the control sequence is a promoter comprising SEQ ID NO: 4～7,53,55,57,59,61,67,73,74,76 or 7 nucleotide sequence or a fragment or variant thereof represented by 8 including.

In a preferred embodiment, the regulatory sequence comprises a terminator comprising the nucleotide sequence set forth in SEQ ID NOs: 8-11, 54, 56, 58, 60, 62, 102 , 104 , 107 , or 108 , or fragments or variants thereof. Including.

In certain particularly preferred embodiments, the recombinant gene construct of this aspect comprises a flanking sequence of or surrounding a nucleic acid fragment that can be inserted into the genetic material of a plant. In some embodiments, the flanking sequences, or portions thereof, are derived from one or more plants. Preferably, the flanking sequence includes a restriction digestion site. In certain particularly preferred embodiments, one or more of the flanking sequences is a nucleotide sequence set forth in SEQ ID NO: 98 , 99 , 105 , 106 , 111 , 112 , 113 , 114 , 116 , or 117 , or a fragment or mutation thereof. Including the body.

Preferably, the flanking sequence comprising the restriction digestion site facilitates the removal or excision of one or more fragments of this aspect of the recombinant gene construct consisting of larger scale constructs and / or plant derived sequences from the vector. . With reference to the embodiment, as an example, a preferred gene construct of SEQ ID NO: 73～74,78, and 7 9, the flanking sequences so as to facilitate removal of fragments of recombinant gene construct comprising a plant-derived sequences It will be understood to include.

In certain other preferred embodiments, the selectable marker sequence facilitates selection by conferring salt tolerance on genetically improved plants. In a preferred embodiment, the selectable marker comprises the nucleotide sequence shown in SEQ ID NO: 115 belonging to the DREB1A gene as described above.

The backbone sequence of this vector is that of the vector pKannibal. It contains the promoter sequence of the sorghum biocolor UBIQUITIN1 gene (Sobic.004G049900) and the terminator of the sorghum biocolor UBIQUITIN2 gene (Sobic.004G050000). It consists of primer F 5 ′ Phos cctcacGTGTTACACAGCTCAATTTACAGACTACTCACC (SEQ ID NO: 123 ) (3 nucleotides at the start of the promoter to create a blunt cutter site PmlI to allow excision of the intragenic cassette prior to direct gene transfer. And the natural PstI site at the 3 ′ end of the Ubi1 promoter amplified from sorghum gDNA using R tccCTGCAGAAGTCACCAAAAATAGGGT (SEQ ID NO: 122 ). The fragment was digested with PstI and ligated into the vector pKannibal excised with StuI and PstI. Terminator Ubi1, (to create a blunt cutter cloning site Sfol, adding 3 nucleotides to the start site of the terminator) Primer F TishishishitijishieijishijishitieijigcGCCATAGGTCGTTTAAGCTGCTG (SEQ ID NO: 124) and R TishishishieishitieijitishieishijitiGTATAGCACAATGCATGATCTTGCT (SEQ ID NO: 125) (for excision of the gene cassette In order to create a blunt cutter site PmlI and a SpeI site for insertion into the preceding vector, 3 nucleotides were added to the end of the terminator). The fragment was digested with PstI and SpeI and ligated into two previously obtained intermediate vectors excised with the same enzymes.

A schematic diagram of another preferred gene construct (pSbiUbi2) is shown in FIG. The complete nucleotide sequence of this gene construct is shown in SEQ ID NO: 74.
The backbone sequence of this vector is that of the vector pKannibal. It contains the promoter and terminator sequences of the sorghum biocolor UBIQUITIN2 gene (Sobic. 004G050000). It consists of primers F 5Phos / cctcacGTGAGGCCCCTATAGATGTTAGTAAATATAGCTAAA (SEQ ID NO: 126 ) (adding 3 nucleotides to the promoter start site to create a blunt cutter site PmlI to allow for excision of the intragenic cassette) and R tccCTGCAGAAGAGTCAGGATA sequence 127 ) was used to exploit the native PstI site at the 3 ′ end of the Ubi2 promoter amplified from sorghum gDNA. The fragment was digested with PstI and ligated into the vector pKannibal digested with StuI and PstI. Terminator Ubi1 was amplified and cloned as described above for pSbiUbi1.

A schematic diagram of such a double T-DNA vector containing the gene construct is shown in FIG. The sequence of this vector is shown in SEQ ID NO: 70.
Apart from the selectable marker gene (nptII), a visual marker gene (ANT1) for anthocyanin biosynthesis is included to allow easy outcrossing. The vector structure was as follows. That is, using T-DNA containing the tomato PARTIAL ACTIN promoter and terminator, a blank pIntrA cloning vector as template, the forward primer (BsiWI) CGTACGGAATGCCAGCACTCC (SEQ ID NO: 128) and reverse primer (BsrGI) TijitieishieieitishijitiCAACGTTCACTTCTAAAGAAATAGC (SEQ ID NO: 129) And was inserted into a single T-DNA plasmid by digestion with BsiWI enzyme (pArt27 RbcS3C: ANT1: RbcS3C 35S: nptII: NOS).

In order to transform plants, both shown amiRNA based approaches (amiRNA10 and amiRNA11) were combined in one fully in-gene construct. As shown in FIG. 35 and SEQ ID NO: 81, a vector of “2 Vector 2 Agrobacterium strain co-transformation method” was generated using pArt27 as a backbone that can be used in combination with another selectable marker construct. However, it can be outbred at a later stage prior to commercialization. For this purpose, the sequence (SEQ ID NO: 81) was inserted into pIntrA (FIG. 18; SEQ ID NO: 67). First, SEQ ID NO: 81 is synthesized, and then F primer 5 ′ Phos
Amplified with GATTAAAAAGAGCAGGGAAAGTATTGGGGTGGAGAATTTG (SEQ ID NO: 130 ) and R primer 5 'Phos CcgaagagagtgaaggtgaTGATCA (SEQ ID NO: 131 ), excised with ACTin promoter and terminator, and complemented with p. The orientation of the insert was tested by sequencing.

Alternatively, the double cassette (one vector containing two T-DNA cassettes) shown in FIG. 20 and SEQ ID NO: 70 was used. For this purpose, a double amiRNA T-DNA cassette (SEQ ID NO: 81) was inserted into pArt27 RbcS3C: ANT1: RbcS3C 35S: nptII: NOS (FIG. 19; SEQ ID NO: 69). First, a double amiRNA T-DNA was amplified from the vector shown in FIG. 35 using primers: forward (BsiWI) CGTACCGGAATGCCAGCACTCC (SEQ ID NO: 132 ) and reverse (BsrGI) TGTACAATCGTCAACGTTCACTTCTAAAGAAAATAGC (SEQ ID NO: 133 ); It was inserted into a single T-DNA plasmid pArt27RbcS3C: ANT1: RbcS3C 35S: nptII: NOS (FIG. 19; SEQ ID NO: 69) excised with the BsiWI enzyme. Tomato plant transformation, regeneration and CMV loading experiments in this manner are currently underway. The genetic organization and complete sequence of this duplex T-DNA vector for durable intragenic CMV resistance is shown in FIG. 36 and SEQ ID NO: 82.

In order to transform plants, the sequence (SEQ ID NO: 83) was inserted into pIntrA (FIG. 18; SEQ ID NO: 67). First, SEQ ID NO: 83 was synthesized, and then amplified using F primer 5 ′ Phos GATTAAAAAGAGCGAGAAAGTATGGGTGGAGAATTTG (SEQ ID NO: 134 ) and R primer 5 ′ Phos CcgaagagagtgaaggtgaTGATCA (SEQ ID NO: 135 ), and the complement promoter of ACTIN was amplified. Thereafter, it was ligated with pIntrA excised with HpaI and PmlI. The orientation of the insert was tested by sequencing.

In order to develop this approach for multiple virus resistance in sorghum, amiRNA was designed to target either multiple viruses or multiple virus isolates of the same virus. Initially, a sufficiently long intragenic sequence was identified that matched JGMV, SCMV and / or MDMV within the conserved region. The nucleotides in the natural sorghum microRNA Sbi-miR156b were then replaced with various intragenic antiviral amiRNA sequences. Some of these are shown in FIGS. 38-39 and SEQ ID NOs: 83-89. AmiRNA was synthesized and amplified using primers F tccCTGCAGgcactttgcctgaagagagaggacg (SEQ ID NO: 136 ) and R 5 ′ Phos gctccaaaatcgagagagatagacc (SEQ ID NO: 137 ); pst I and digested with p 73) or pSbiUbi2 (FIG. 22; SEQ ID NO: 74). The obtained plasmid was cleaved with PmlI to obtain a minimal intragenic transformation cassette.

Cloning methods for these constructs are as follows. AmiRNA4 was obtained by using primers F tccCTGCAGgcactttgcctgaagagagaggac (SEQ ID NO: 138 ) (PstI site added to the 5 ′ end) and R gtgactactagaggagagagagagagcccc (SEQ ID NO: 139 ) (ApaL added to the 3 ′ site). AmiRNA5 used primers Fgtgcactttgcctgaagagagaggac (SEQ ID NO: 140 ) (ApaLI site added to the 5 ′ end) and R aacccctagggctccaaatcggagagatagag (SEQ ID NO: 141 ) added to the 3 ′ end (AvrII site added). AmiRNA2 was amplified using primers F cctagggggttttgcactttgcctg (SEQ ID NO: 142 ) (AvrII site added to the 5 ′ end) and R 5 ′ Phos gctccaaattcgacagagatagagc (SEQ ID NO: 143 ). The fragment was digested with each enzyme and ligated into either vector pSbiUbi1 or pSbiUbi2 excised with PstI and SfoI in a single reaction.

In order to develop an intragenic amiRNA approach for virus resistance in rice, the amiRNA targets the helper virus, Rice tungro spurious virus (RTSV), that mediates the severity of symptoms caused by RTBV Designed. For this purpose, nucleotides in the native rice microRNA Osa-miR156a were replaced with various intragenic antiviral amiRNA sequences. One of these (amiRNA1) is shown in FIG. 42 and SEQ ID NO: 93. To create an in-gene rice transformation cassette, the amiRNA1 sequence is converted into primers FGAGCtcaatgttatctatacccatgcacatatgg (SEQ ID NO: 144 ) (introducing nucleotides to complete the complete SacI site to its 5 'end) and the R 5'Phostaggatagtattagtagt No. 145 ) was synthesized and amplified. It was restricted with SacI and inserted into pOsaAPX (FIG. 23; SEQ ID NO: 76) excised with SacI and blunt end cutter PsiI, whose three final nucleotides are identical to its full sequence in the database, native Osa-miR156a This contributes to the “continuity” of folding. Further testing on rice plants is currently underway.

To test whether may result in structural trait of desirable plant regulatory other Mediator subunits in tomato, the MED25 Orusoro grayed putative identified in tomato, intragenic AmiRNA (SEQ ID NO: 96; Figure 50) Is designed for its downward control. As shown in FIG. 50, when using the double luciferase assay in N. benthamiana above, amiRNA6 significantly down-regulates the tomato MED25 sequence (P <0.001; Student t-test). I was able to. AmiRNA9 was inserted into pIntrA and tomato plants were transformed as described above. Nine PCR positive transformants (lines) were tested for expression of amiRNA6 and knockdown of MED25 using qRT-PCR. As shown in FIG. 50, all nine lines expressed amiRNA6, and the expression of MED25 was significantly (P <0.05) lower in all generated lines as compared to the wild type plant.

In addition, in order to improve the nutritional value of the main commercially consumed food crops that are widely consumed, a new Reiziq rice cultivar plant was created that harbors a completely in-gene cassette to increase the anthocyanin levels in the rice grain. Rice cultivars Reiziq plants, ACTIN1: DREB1A: a flanked rice OSB2 gene by natural R1G1B promoter and terminator in addition to DREB1A cassette with including gene in construct was transformed as described above. Prior to the particle gun, the intragenic OSB2 cassette was excised and purified by cutting with FspI and ApalI restriction enzymes. The rice R1G1B promoter and terminator cassette were selected because the corresponding genes are most strongly expressed in mature rice grain endosperm. The plant has been skillfully produced as shown in FIG. 51, but currently grows to a maturity level that only measures the anthocyanin level in the rice grain. The consumer acceptability of these plants is expected to increase as these plants provide direct consumer benefits and are completely intragenic. In addition, they are likely to present improved abiotic stress tolerance mediated by an intragenic DREB1A cassette that may benefit growers of this variant. For future crossing with other varieties, it can be expected that these plants will be incorporated into the breeding program.

To create an intragenic RNAi construct in the ACTIN promoter-terminator expression cassette (pIntraA), first add a long “forward” fragment, F primer 5′PhosGATTAAATACAAATCGATCTCATTTCCTCCATC (SEQ ID NO: 146 ) and a temporary complement to the end of the ACTIN promoter Amplification was performed using the R primer tcccaTTGTTCAGTTGAAACAATTTTTTTGTCATATAAC (SEQ ID NO: 147 ) which adds three nucleotides to create an MfeI restriction enzyme site. The shorter “reverse” fragment is an F primer that adds three nucleotides to create a temporary MfeI restriction enzyme site tcccaTTGGAAGTGTATGAGTTTACAAAACATACTTACCT (SEQ ID NO: 148 ) and an R primer that complements the start site of the ACTIN terminator 5′PhosCTACATATCTCT No. 149 ). The fragment was restricted with MfeI and constructed with a single ligation using pIntrA excised with HpaI and PmlI. When ligating the MfeI site between the long and short fragments, half of them belong to the long fragment and the other half belong to the short fragment. The orientation of the insert was verified for the complementarity of the promoter and terminator. The complete intragenic construct encompassing the LB and RB fragments is shown in SEQ ID NO: 97 and FIG.

Tomato transformation (cv. Moneymaker) is performed by cotransforming the construct of SEQ ID NO: 97 with a marker gene cassette containing both ANT1 and NPTII genes for selection of transformed plants as described in the above example. did. Purple plants (indicating their positive transformation status) were selected and further tested for gene expression by qRT-PCR. Other plants without ANT1 gene expression were also selected. The tomato fruits produced by these plants are each expected to have a pointed, heart-shaped appearance in either purple or red fruit color.

Cultivar Japonica Reiziq with high yield potential is popular among growers but typically lacks the scent found in jasmine (scented) rice. The scent in rice can be obtained by disrupting the expression of the BADH2 gene in rice. Therefore, a BADH2 RNAi cassette with an endogenous R1G1B promoter and terminator expressed in rice endosperm was constructed . Excision of the DNA cassette prior to microprojectile bombardment Lee Nekarusu is achieved using FspI restriction enzymes and agarose gel electrophoresis size fragmentation. An in-gene rice plant under development with a potential scent is shown in FIG.

Claims (23)

One or more nucleic acid fragments adapted for insertion into the genetic material of a plant for altering or modifying a plant trait selected from the group consisting of disease resistance traits, abiotic stress tolerance traits, and morphological traits. A recombinant gene construct comprising, each of said one or more nucleic acid fragments consisting of a plurality of nucleotide sequences of at least 15 nucleotides length from one or more plants, wherein said one or more of the plant genetic material comprises The recombinant gene construct, wherein the nucleotide sequence inserted into the genetic material of the plant comprises a nucleotide sequence derived from the plant when the nucleic acid fragment of 1. is inserted. The recombinant gene construct according to claim 1, wherein the nucleotide sequence derived from one or more plants is (i) derived from one plant or (ii) derived from a plurality of plants of the same species . The total length of said one or more nucleic acid fragments of said genetic construct that is insertable into the genetic material of a plant is at least 100 base pairs; at least 500 base pairs; at least 1000; at least 2000 base pairs; or at least 3000 base pairs, The recombinant gene construct according to claim 1 or 2 . Said one or more nucleic acid fragments of the gene construct can be inserted into the genetic material of the plant, comprising a nucleotide sequence for one or more expression in plants, the set according to any one of claims 1 to 3 Altered gene construct. 5. The recombinant gene construct according to claim 4 , wherein the one or more nucleotide sequences for expression in a plant are adapted for expression in the plant to alter or modify traits of the plant. 6. The recombinant gene construct according to claim 4 or 5 , wherein the nucleotide sequence for expression in plants comprises a nucleotide sequence encoding a protein. 7. The recombinant gene construct of claim 6 , wherein the non-protein coding nucleotide sequence comprises one or more small RNA nucleotide sequences. The recombinant gene construct according to any one of claims 8 to 9 , wherein the one or more nucleotide sequences for expression in plants comprises one or more selectable marker nucleotide sequences. The recombinant genetic construct according to any one of claims 1 to 9 , wherein the one or more nucleic acid fragments of the genetic construct that are insertable into the genetic material of a plant include one or more regulatory nucleotide sequences. Further comprising flanking sequences or flanking sequences surrounding them insertable said one or more nucleic acid fragments on the genetic material of a plant, recombinant gene construct according to any one of claims 1-9. First border nucleotide sequence;
A second border nucleotide sequence; and one or more additional nucleotide sequences located between said first border nucleotide sequence and said second border nucleotide sequence, wherein said additional nucleotide sequence and said At least a portion of the additional nucleotide sequence to adjacent said first boundary nucleotide sequences are derived from one or more plants, recombinant gene construct according to any of claims 1-10. 12. The recombinant gene construct of claim 11 , wherein the first border sequence belongs to the right border nucleotide sequence functional in Agrobacterium-mediated T-DNA plant transformation. 13. The genetic construct according to claim 11 or 12 , wherein the second border nucleotide sequence belongs to the left border nucleotide sequence that is functional in Agrobacterium-mediated T-DNA plant transformation. Nucleotide sequences represented by SEQ ID NOs: 1-35, 49, 51-56, 66-68, 71-92, or 94-97, or nucleic acids encoding the amino acid sequences represented by SEQ ID NOs: 38-46, or fragments thereof. include variants, recombinant gene construct according to any one of claims 1 to 13. A method for producing a recombinant gene construct, comprising a plant gene for modifying or modifying a plant trait selected from the group consisting of disease resistance traits, abiotic stress resistance traits, and morphological traits Deriving from the one or more plants one or more nucleic acid fragments adapted for insertion into the material, wherein the one or more nucleic acid fragments consist of a plurality of nucleotide sequences of at least 15 nucleotides in length, When the recombinant gene construct is produced by the method described above and the one or more nucleic acid fragments are inserted into the genetic material of the plant, the nucleotide sequence inserted into the genetic material of the plant consists of the nucleotide sequence derived from the plant. ,Method. Adding a first border nucleotide sequence and a second border nucleotide sequence to each end of the one or more additional nucleotide sequences, wherein the one or more additional nucleotide sequences and the first border nucleotides 16. The method of claim 15 , wherein at least a portion of the sequence is from one or more plants. A recombinant gene construct produced by the method of claim 15 or 16 . A vector comprising the recombinant gene construct according to any one of claims 1 to 14 and a backbone nucleotide sequence. A host cell comprising the gene construct according to any one of claims 1 to 14 or the vector according to claim 18 . A method of genetically improving a plant comprising the step of inserting at least one nucleic acid fragment of the genetic construct according to any one of claims 1 to 14 and 17 into the genetic material of a plant cell or plant tissue. , Wherein the plant to be genetically improved belongs to the same species of the one or more plants from which the one or more nucleotide sequences of the nucleic acid fragment of the genetic construct are derived. The at least one nucleic acid fragment of the genetic construct is (i) via Agrobacterium-mediated transformation of the plant cell or plant tissue, or (ii) via direct transformation; 21. The method of claim 20 , wherein the method is inserted into the genetic material of cell or plant tissue. A plant or plant part made according to the method of claim 20 or 21 . A plant, wherein at least one nucleic acid fragment of a recombinant gene construct has been inserted into the genetic material of said plant, wherein said recombinant gene construct has a disease resistance trait, an abiotic stress resistance trait, and a morphological trait. Comprising one or more nucleic acid fragments adapted for insertion into the genetic material of the plant for altering or modifying the traits of the plant selected from the group consisting of: And a nucleotide sequence inserted into the genetic material of the plant, the nucleotide sequence inserted into the genetic material of the plant is A plant consisting of a derived nucleotide sequence.